Sialic acid-binding Ig-like lectin-7 (Siglec-7), is a type 1, immunoglobulin-like, transmembrane protein expressed on immune and hematopoietic cells, including immature and mature myeloid cells, such as monocytes, macrophages, dendritic cells, neutrophils, mast cells, and microglial cells, as well as lymphoid cells, such as natural killer cells, and subsets of T cells (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; Angata and Varki (2000) Glycobiology 10:4: 431-438, Nicoll et al (1999) JBC 274:48: 34089-34095; Falco et al. (1999) J. Exp. Med. 190: 793-802). Siglec-7 is a member of the Siglec family of lectins that bind sialic acid residues of glycoproteins and glycolipids. One potential binding target for Siglec proteins are gangliosides; that is, glycolipids that consist of a ceramide linked to a sialylated glycan. Most gangliosides share a common lacto-ceramide core and one or more sialic acid residues. Diversity in the Siglec ligands is generated by the addition of other neutral sugars and sialic acid in different linkages, and modification of sialic acid itself.
Fourteen Siglec proteins have been identified in humans and nine in mice that are comprised of 2-17 extracellular Ig domains including an amino-terminal V-set domain that contains the sialic acid-binding site. The sialic acid-binding region is located on the V-set Ig-like domain, which contains a two aromatic residues and one arginine motif highly conserved in all Siglecs (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; Von Gunten and Bochner (2008) Ann NY Acad Sci. 1143:61-82; May et al. (1998) Mol Cell. 1:719-728; Crocker et al. (1999) Biochem J. 341:355-361; and Crocker and Varki (2001) Trends Immunol. 2:337-342). The binding sites to sialylated ligands have been mapped by crystal structures with and without ligand bound (Alphey et al., (2003) J. Biol. Chem. 278:5: 3372-3377; Attrill et al., (2006) J. Biol. Chem. 281 32774-32783; and Varki et al., Glycobiology, 16 pp. 1R-27R). Since cell membranes are rich in sialic acids, ligand binding by Siglecs can occur in cis and in trans, both affecting their functional properties. Each Siglec has a distinct preference for binding the diverse types of sialylated glycans that are found on the surface of mammalian cells (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; and Crocker et al. (2007) Nat Rev Immunol. 7:255-266). Most Siglec proteins, including Siglec-7, contain one or more immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in their cytoplasmic tails, which enable them as inhibitory receptors and negative regulators of immune functions through recruitment of the tyrosine phosphatases SHP1 and SHP2 (Crocker et al. (2007) Nat Rev Immunol. 7:255-266; McMillan and Crocker (2008) Carbohydr Res. 343:2050-2056; and Von Gunten and Bochner (2008) Ann NY Acad Sci. 1143:61-82). Certain Siglecs contain immunoreceptor tyrosine-based activating motif (ITAM) sequences in their cytoplasmic tails, which enable them to act as activating receptors and positive regulators of immune function through predicted recruitment of spleen tyrosine kinase (Syk) (Macauley S M. et al., (2014) Nature Reviews Immunology 14, 653-666). The Siglec protein family is associated with multiple human disease including, autoimmunity, susceptibility to infection, multiple types of cancer including lymphoma, leukemia and acute myeloid leukemia, systemic lupus erythematosus, rheumatoid arthritis, neurodegenerative disorders, asthma, allergy, sepsis, chronic obstructive pulmonary disease, graft-versus-host disease, eosinophilia, and osteoporosis (Macauley S M. et al., (2014) Nature Reviews Immunology 14, 653-666).
Siglec-7 was cloned in 1999 (Falco et al. (1999) J. Exp. Med. 190: 793-802; Nicoll et al (1999) JBC 274:48: 34089-34095; Angata and Varki (2000) Glycobiology 10:4: 431-438), and selective expression was detected on granulocytes, monocytes, resting and activated natural killer cells and a subset of resting CD8+ T cells in human peripheral blood (Nicoll et al (1999) JBC 274:48: 34089-34095; Falco et al. (1999) J. Exp. Med. 190: 793-802).
Siglec-7 contains an extracellular N-terminal Ig-like (immunoglobulin-like) V-type domain, two Ig-like C2-set domains as well as one consensus ITIM motif and a non-conforming membrane-distal ITIM-like motif in its cytoplasmic domain. Siglec-7 was shown to bind red blood cells in a sialic acid dependent manner due to loss of binding upon sialidase treatment. The binding is thought to be mediated by α2-3 or α2-6 sialic acid linkages (Nicoll et al (1999) JBC 274:48: 34089-34095; Angata and Varki (2000) Glycobiology 10:4: 431-438). Further investigation revealed that Siglec-7 more potently binds α2-8 disialyl residues with 10 nM affinity and demonstrates higher affinity for branched α2-6 sialyl residues compared to terminal α2-3 or α2-6 sialic acids (Yamaji (2002) J. Biol. Chem. 277:8 6324-6332). In vivo Siglec-7 ligands are expressed on b-series gangliosides such as GD2, GD3, and GT1b, which can be found on cells of the central nervous system, melanoma cells, and subsets of T cells (Urmacher et al. (1989) Am. J. Dermatopathol. 11: 577-581, Kniep et al. (1993) Blood 82: 1776-1786). High resolution crystal structure of the N-terminal V-set Ig-like domain of Siglec-7 suggests that ligand binding specificity of Siglec family members resides in the variable C-C′ loop (Alphey et al. (2003) J. Biol. Chem 278:5 3372-3377).
Siglec-7 undergoes phosphorylation of Tyr-437, and Tyr-460 by tyrosine kinases, likely c-Src or Lck (Avril et al., (2004) J. Imm 173: 6841-6849). Following phosphorylation predominantly on the proximal Tyr-437, but also on distal Tyr-460 of its ITIM domains, Siglec-7 binds SHP-2/PTPN11 and SHP-1/PTPN6 (Avril et al., (2004) J. Imm 173: 6841-6849). Phosphatase activity is associated with decreased intracellular calcium mobilization, and decreased tyrosine phosphorylation on multiple proteins (Ulyanova, T., et al., (1999) Eur J Immunol 29, 3440-3449; Paul, S. P., et al., (2000). Blood 96, 483-490) as well as with blockade of signal transduction and immune response, in part, through dephosphorylation of signaling molecules on adjacent activating receptors, including those that contain ITAM motifs, pattern recognition receptors, Toll-like receptors and damage-associated molecular pattern (DAMP) receptors.
Some, but not all, Siglec ligands induce receptor downregulation (Macauley S M. et al., (2014) Nature Reviews Immunology 14, 653-666). Ligand-induced receptor degradation has been reported for tyrosine kinase receptors (Monsonego-Oran et al., (2002) Febs letters 528, 83-89; and Fasen et al., (2008) Cell & Molecular Biology 9. 251-266), as well as steroid receptors (Callige et al., (2005) Mol. Cell. Biol. 25. 4349-4358; and Pollenz et al., (2006) Chemico-Biological Interactions. 164. 49-59). Suppressor of cytokine signaling 3 (SOCS3) has been shown to compete with SHP-1/2 and binds Siglec-7 upon ITIM phosphorylation in the presence of Siglec-7 crosslinking (Orr et al. (2007) J. Biol. Chem. 282: 3418-3422). SOCS3 binding results in ECS E3 ligase targeting of Siglec-7 for proteasomeal degradation (Willams and Palmer (2012) Biochem. Soc. Trans. 40: 215-218; Orr et al. (2007) J. Biol. Chem. 282: 3418-3422).
Activation of Siglec-7 signaling has also been shown to be associated with an increase in production of proinflammatory cytokines IL-6, IL-1alpha, MIP-1beta, IL-8 and TNFalpha, as well as upregulation of adhesion molecules ICAM-1 and CD49e selectively in human monocytes (Varchetta et al. (2012) PLOS One 7: 9: e45821). These activities of Siglec-7 appear to be mediated through the phosphorylation of ERK (Varchetta et al. (2012) PLOS One 7: 9: e45821). It has been proposed that the association between ITIM-containing Siglec receptors and activating receptors may be mediated by extracellular ligands that bind and bridge these receptors (Macauley S M. et al., (2014) Nature Reviews Immunology 14, 653-666).
Multiple studies indicate an inhibitory role for Siglec-7 in function of natural killer cells, regulation of T cell receptor signaling, and attenuation of signaling in DCs (Crocker et al., (2012) Ann. N Y Acad. Sci. 1253, 102-111; Pillai et al., (2012) Annu. Rev. Immunol. 30, 357-392; von Gunten and Bochner (2008) Ann. N Y Acad. Sci. 1143, 61-82; Ikehara et al. (2004) J. Biol. Chem. 279:41 43117-43125; Nicoll et al. (2003) Eur. J. Imm 33:6:1642-1648; Hudak et al. (2013) Nat. Chem. Biol.; Bax et al. (2007) J. Imm 179: 12: 8216-8224; Lock et al. (2004) Immunobiology 209: 1-2:199-207). Functional studies in natural killer cells have demonstrated that tumor cells expressing Siglec-7 binding sialic acid ligands inhibit NK cell activation and tumor cell killing. Many human tumors robustly upregulate sialic acid ligands, which enables immune evasion and cancer progression (Jandus et al. (2014) J. Clinic. Invest. 124:4: 1810-1820). Moreover, Hudak et al. performed glycocalyx engineering and showed that cells coated with synthetic sialoside glycopolymers were protected from NK cytotoxicity. It is proposed that sialic acid upregulation on tumors facilitates a state of “super self” that strongly inhibits natural killer cell immunosurveillance (Macauley and Paulson (2014) Nat. Chem. Biol. 10:1: 7-8).
There is no apparent mouse homolog of Siglec-7; however mouse Siglec-E is 53% similar, therefore the closest related Siglec. In mice, genetic inactivation of Siglec-E does not lead to obvious developmental, histological, or behavioral abnormalities; and Siglec-E-deficient mice breed normally, indicating that Siglec-E is not an essential gene and that its function may be limited to innate immunity (McMillan et al. (2013) Blood 121:11: 2084-2094). Upon challenge of Siglec-E deficient mice with aerosol LPS, increased neutrophil recruitment in the lung was demonstrated, which could be reversed by blockade of the β2-integrin CD11b. The Siglec-E deficient neutrophils were shown to have increased phosphorylation of Syk and p38 MAPK in a CD11b-dependent manner. This data suggests that Siglec-E functions to suppress neutrophil recruitment in a model of acute lung inflammation (McMillan et al. (2013) Blood 121:11: 2084-2094).
In oncology, Siglec-7 has been suggested as a therapeutic target for chronic and acute myeloid leukemic as crosslinking Siglec-7 inhibited cellular proliferation (Vitale et al. (1999) PNAS 96: 15091-15096; Vitale et al. (2001) PNAS 98:10: 5764-5769). Siglec-7 activity has also been shown to inhibit cytokine-induced cellular proliferation (Orr et al. (2007) J. Biol. Chem. 282: 3418-3422).
Antibodies to Siglec-7 have been described in, for example, WO2011038301, Jandus et al. (2014) J. Clinical Invest. 124:4: 1810-1820, Varchetta et al. (2012) PLOS One 7: 9: e45821 et al. (2012). Falco et al. (1999) J. Exp. Med. 190: 793-802, Nicoll et al (1999) JBC 274:48: 34089-34095, Nicoll et al. (2003) Eur. J. Imm 33: 1642-1648. However, these antibodies do not display the functional characteristics required for a therapeutic antibody.
Accordingly, there is a need for therapeutic antibodies that specifically bind Siglec-7 and reduce Siglec-7 expression on the cell surface, reduce interactions between Siglec-7 and one or more Siglec-7 ligands, and/or reduce one or more Siglec-7 activities in order to treat one or more diseases, disorders, and conditions associated with undesired Siglec-7 activity.
All references cited herein, including patents, patent applications and publications, are hereby incorporated by reference in their entirety.